The overall objective of this proposal is to help elucidate the mechanism of action of the glycosidases. To this end it is proposed to isolate the active-site peptide from a labeled glucosyl-enzyme complex by the phenol-quenched reaction of pure yeast alpha-methyl-D-glucosidase with C14- methyl-alpha-D-glucoside. It is planned to identify in this peptide the glycosylated amino acid, establish the chirality of the glucosidic bond and ascertain the sequence of the adjoining amino acids. In order to see what features of the active peptide are unique to glycosidases, similar studies will be carried out with a second available pure glucosidase, yeast maltase, as well as with glycosidases of various specificities, including a number of medical physiological significance, specifically those of lysosomal and intestinal origin. Kinetic procedures will be used to characterize the ionizable groups of the enzyme. Glycosylamines, which we have shown to be specific competitive inhibitors will be synthesized in different basicities and used to probe the active center of the glucosidases. The effect of specific functional group modification of pure maltase and alpha-methylglucosidase on substrate binding and other kinetic parameters will be studied in an effort to implicate participating amino acid residues. From the results obtained from the several lines of approach, an effort will be made to explain the reaction mechanism of alpha-methyl glucosidase and maltase, in particular, and glycosidases in general with the view of establishing a basis for understanding and controlling glycosidases of importance in physiology.